液相色谱-串联质谱法同时测定大鼠血浆中咪达唑仑和非那西丁及其药动学研究

Simultaneous determination and pharmacokinetics of midazolam and phenacetin in rat plasma by LC-MS/MS

目的建立液相色谱-串联质谱法（LC-MS/MS）同时测定非那西丁（PN）、咪达唑仑（MDZ）在大鼠血液中的含量。方法大鼠随机被分为PN组、MDZ组和PN-MDZ合用组（均n=6）,分别尾静脉注射PN、MDZ及PN和MDZ混合探针药物,剂量均为1 mg·kg-1。于指定时间眼眶采集血样,以苯海拉明为内标,采用LC-MS/MS测定大鼠血浆中探针药物及其代谢产物的浓度。色谱柱为Kinetex XB-C18柱（100 mm×3.0 mm,2.6μm）,流动相为甲醇∶0.025%甲酸水,进行梯度洗脱。电喷雾离子源,以多反应离子监测方法进行正离子扫描,PN和其代谢产物醋氨酚（Ace）,MDZ和其代谢产物1-羟基咪达唑仑（1-OH-MDZ）及苯海拉明离子对分别为m/z 180.2→110.0,m/z 152.2→110.1,m/z 326.2→291.2,m/z 342.2→324.2和m/z 256.3→167.2。结果PN、Ace、MDZ和1-OH-MDZ线性范围分别为：4.288~21 440 ng·m L-1、1.038~5 190 ng·m L-1、4.664~11 660 ng·m L-1、0.01~50 ng·m L-1;回收率、稳定性和日内、日间精密度均符合生物样品分析要求;PN和MDZ单用与合用前后药动学参数无显著差异（P〉0.05）。PN单用和与MDZ合用后t1/2分别为（0.44±0.15）、（0.42±0.08）h,ρmax分别为（9.35±1.58）、（10.17±0.76）μg·m L-1,AUC0-6 h分别为（4.21±0.63）、（4.90±0.42）μg·h·m L-1;MDZ单用和与PN合用后t1/2分别为（0.64±0.09）、（0.68±0.05）h,ρmax分别为（3.48±0.51）、（3.01±0.64）μg·m L-1,AUC0-6 h分别为（2.58±0.41）、（2.08±0.29）μg·h·m L-1。结论建立的测定方法可用于PN、MDZ以及其代谢产物的药动学研究,并证明PN、MDZ在大鼠体内基本没有代谢的相互作用。

AIM To establish a LC-MS/MS method for the simultaneous determination of phenacetin（ PN） and midazolam（ MDZ） in rat plasma. METHODS Rats were divided into PN, MDZ, PN and MDZ combination groups（ n = 6 in each）. Blood samples were collected from the eyes at specified times after phenacetin（ 1 mg·kg-1）, midazolam（ 1 mg·kg-1） and phenacetin（ 1 mg·kg-1） ＋ midazolam（ 1 mg·kg-1）mixed probe drug solutions were given to rats individually by intravenous injection. The plasma concentration of PN, MDZ and their metabolites were detected by LC-MS/MS, in which diphenhydramine was used as the internal standard（IS）. The analytes were separated using a Kinetex XB-C18（100 mm × 3.0 mm, 2.6 μm）.The mobile phase consisted of methanol-0.025% formic acid water solution with a gradient elution. Electrospray ionization source was applied, the protonated ion of the analytes was detected in positive ionization by multiple reaction monitoring mode. The mass transition pairs of m/z 180.2 →110.0 for PN, m/z 152.2 →110.1 for acetaminophen, m/z 326.2→291.2 for MDA, m/z 342.2→324.2 for 1-hydroxymidazolam and m/z 256.3→167.2for IS. RESULTS The linear calibration curve of PN, acetaminophen, MDZ and 1-hydroxymidazolam were obtained in the concentration range of 4.288-21 440 ng·m L-1, 1.038-5 190 ng·m L-1, 4.664-11 660 ng·m L-1and 0.01-50 ng·m L-1respectively. Recoveries, stabilities, the intra-day and inter-day precisions were all met the requirements of biological samples analysis. The pharmacokinetic parameters of PN and MDZ had no significant change after combination administration（P 〉 0.05）. The t1/2of PN administrated alone and combined with MDZ were（0.44 ± 0.15） and（0.42 ± 0.08） h, ρmaxwere（9.35 ± 1.58） and（10.17 ± 0.76） μg·m L-1,AUC0-6 hwere（4.21 ± 0.63） and（4.90 ± 0.42） μg·h·m L-1. The t1/2of MDZ administrated alone and combined with PN were（ 0.64 ± 0.09） and（ 0.68 ± 0.05） h, ρmaxwere（ 3.48 ± 0.51） and（ 3.01 ± 0.64） μg·m L-1,AUC0-6 hwere（ 2.58 ± 0.41） and（ 2.08 ± 0.29） μg·h·m L-1. CONCLUSION The method is successfully applied to pharmacokinetic study of PN, MDZ and their metabolites. The results show there is no metabolic interaction between PN and MDZ in vivo.